The service life of chromatographic column is closely related to the daily maintenance besides the analysis of sample, mobile phase and service frequency. The service life of chromatographic column is mainly measured by column efficiency and column pressure. If the column efficiency is too low or the column pressure is too high, it is generally considered that the chromatographic column has ended. Therefore, the key to prolong the service life of chromatographic column is to eliminate the factors that cause the decrease of column efficiency and the increase of column pressure. The following is the routine maintenance method of chromatographic column:

 

1、 The pH of mobile phase shall be within the range of use

 

If the mobile phase exceeds its pH range, it will lead to the loss of silica matrix and the fracture of carbon chain of the bonding phase, which will reduce the column efficiency and shorten the service life. Due to the improper pH control of the mobile phase, the damage to the chromatographic column is usually very difficult to restore the chromatographic column, so we must take it seriously and strictly control the pH value of the mobile phase.

 

2、 Remove solid particles from sample and mobile phase

 

The solid particles contained in the sample and mobile phase will block the sieve plate of chromatographic column, which will not only increase the column pressure, but also decrease the column efficiency, because the blocking of sieve plate will cause the uneven liquid flow, leading to the tailing and broadening of chromatographic peak type, thus reducing the column efficiency. Therefore, it is recommended to use ultrapure water and chromatographic reagent to filter the sample before analysis, and the mobile phase passes through 0.45 μ M filter membrane.

 

3、 Use protective column or on-line filter

 

The sample and mobile phase can not completely eliminate the solid particles after filtration, because the wear of the pump, the aging of the sealing ring and the pipeline will also produce the solid particles. These solid particles are brought into the chromatographic column by the mobile phase, blocking the sieve plate, resulting in the increase of the column pressure and the decrease of the column efficiency. There are sieve plates on both the protection column and the on-line filter, whose pore size is the same as that of the chromatographic column, so it can prevent the solid particles from reaching the chromatographic column and effectively prevent the blockage of the sieve plate of the chromatographic column. Since the rise of column pressure accounts for a large proportion of analysis failures, in addition to filtering the sample and mobile phase, it is recommended that you add a protective column or online filter at the injection end of the chromatographic column.

 

If the column pressure rise is caused by the plugging of the sieve plate at the injection end, the following methods can be selected for remedy:

 

1. Add protective column or on-line filter in front of chromatographic column, and then reverse flush chromatographic column with methanol and water = 20 / 80ml / min for 180min

 

2. Add protective column or on-line filter at the injection end of chromatographic column, and then use it in reverse direction.

 

4、 Proper use of buffer salt

 

Buffer salt is easy to dissolve in water and hard to dissolve in organic solvent, so improper use of buffer salt will make it precipitate, block the micropore and the gap between particles on the filler matrix, make the filler harden and the column pressure rise; at the same time, it will hinder the free expansion of the carbon chain bonded on the matrix, reduce the retention capacity and column efficiency of the chromatographic column. It is very difficult to remove the buffer salt after it is separated out. Therefore, the correct use of buffer salt is very important to prolong the service life of chromatographic column.

 

The purpose of using the buffer salt correctly is to prevent the buffer salt from evaporating out, so the method of using the buffer salt correctly can be summed up as one sentence: filter before use and wash after use. The specific methods are as follows:

 

1. Equivalency condition: before and after use of buffer salt, it is necessary to wash the column for 60min at a flow rate of 1.0ml/min with transitional mobile phase; another method to remove buffer salt after use is to wash the column overnight with transitional mobile phase at a flow rate of 0.2ml/min.

 

2. Gradient condition: before running the gradient with a mobile phase containing buffer salt, wash the column with a flow rate of 1.0ml/min for 60min with a transitional mobile phase of the same composition as the initial mobile phase, and then wash the column with the transitional mobile phase of 1.0ml/min for 120min. The gradient setting of the mobile phase containing buffer salt should be as gentle as possible to avoid buffer salt evolution in the gradient process.

 

Note: the transitional mobile phase refers to the organic phase and water phase with the same composition as the analyzed mobile phase, except that the transitional mobile phase does not contain buffer salt.

 

3. Remedy method of buffered salting out:

 

Scheme 1: reverse flush the column with methanol / 20 / 80 at a flow rate of 1.0ml/min and 35 ° C for 120min

 

Scheme 2: flush the column with methanol / water = 20 / 80 at a flow rate of 0.2ml/min overnight.

 

5、 Prevent retention of strong retention substances on chromatographic column

 

The accumulation of strong retention substances and macromolecular compounds in the chromatographic column results in the extra retention behavior of the compounds in the sample, which not only causes the peak type to be widened and tailed, reduces the column efficiency, but also causes the change of retention time, and when it accumulates to a certain extent, it also causes the column pressure to rise. Since the influence of strong retention substances and macromolecular compounds on chromatographic separation is a cumulative effect, it will take a certain time to reflect it, but for many drugs, especially complex samples, it is difficult to judge whether they contain strong retention substances, so to prevent the accumulation of strong retention substances, it is necessary to clean the chromatographic column with pure methanol or cyanogen in daily maintenance.

 

Cleaning method:

 

1. Unused buffer salt: after the analysis is completed every day, remove the buffer salt with the above method, and then wash the chromatographic column with methanol or cyanogen for 60min.

 

2. Use of over buffered salt: after the analysis, remove the buffered salt with the above method, and then wash the column with pure methanol or cyanogen for 60min.

 

3. Remedies:

 

Water -- cyanogen -- LV imitation (or isopropanol) -- cyanogen -- water

 

Rinse the column with a flow rate of 1.0ml/min for 60min in each step